Transcription-replication collisions trigger high-fidelity replication reset by RecBCD

Double-stranded DNA ends (DSEs), which arise from external damaging agents or intrinsic cellular processes such as transcription-replication collisions (TRCs), pose a threat to genome stability. However, the mechanisms preventing and resolving intrinsic DSE formation remain poorly understood. Here, we present findings from genome-wide CRISPR interference screens designed to uncover factors that protect against DSE formation. To identify these DSE protection factors, we used two screens: one impairing RecA-dependent recombination strand invasion and another restricting RecBCD access to DSEs. Restricting RecBCD access to DSEs uncovered unique gene targets related to ribosome function, absent in repair inhibition-focused screens. We showed that inhibited translation elongation, which disrupts translation-transcription coupling, drives spontaneous formation of DSEs via TRCs. Contrary to previous models, which suggest that TRCs cause double-strand breaks due to replisome collapse, our data imply that replisome stalling followed by collisions between trailing replisomes generate double-strand DNA ends. These DSEs are resolved through a process we term "replication reset," involving degradation of the affected DNA replichore to restore replication fork integrity, without triggering the DNA damage response, or mutagenesis. This study highlights the pivotal role of translation in protecting the genome from TRC-induced stress and reveals an unexpected mechanism for managing intrinsic DNA damage while preserving genome stability.